S254: A NEW GENE AHED PLAYS ESSENTIAL ROLES IN HEMATOPOIESIS THROUGH RNA-SPLICING
Authors: Ritsuko Nakai, Takafumi Yokota, Masahiro Tokunaga, Mikiro Takaishi, Takao Sudo, Henyun Shi, Yoshiaki Yasumizu, Daisuke Okuzaki, Chikara Kokubu, Sachiyo Tanaka, Katsuyoshi Takaoka, Ayako Yamanishi, Junko Yoshida, Hitomi Watanabe, Gen Kondoh, Kyoji Horie, Naoki Hosen, Shigetoshi Sano, Junji Takeda
Journal: HemaSphere (2023)

Background: The development of hematopoiesis in mammals is regulated by coordinated actions of multiple genes. Mutations in some genes are intimately involved in the occurrence of hematological cancers. Given that responsible mutations are yet to be determined in a subset of patients with blood cancers, it is important to identify unknown essential genes in the development of the lympho-hematopoietic system. Aims: We attempted to discover novel functional genes by screening for abnormalities in mutant embryonic stem cells (ESCs) regarding the hematopoietic differentiation potential. Methods: We had previously constructed a homozygous mutant ESC bank by introducing gene-trap insertion vectors and inducing loss-of-heterozygosity in ESCs through transient suppression of the Bloom syndrome gene expression. This mutant ESC bank was used to screen for recessive genetic abnormalities for the development of hematopoietic cells. In vitro differentiation assays of ESCs toward the hematopoietic lineage were performed using a co-culture system with OP9 stromal cells. Results: Among the homozygous mutant ESC clones tested, one clone was severely impaired in terms of differentiation potential toward hematopoietic cells. In this clone, the gene-trap vector was inserted in a previously uncharacterized gene, which locates on mouse chromosome 9, in a biallelic manner. We have named the gene “attenuated hematopoietic development (Ahed)”, according to its function. The gene expression database indicated that the expression level of Ahed was higher in hematopoietic tissues than in other tissues. To understand its specific roles in the hematopoietic system, we generated Ahed conditional knockout (cKO) mice by crossing Ahed-floxed mice with Vav1-cre transgenic mice. We found that fetuses of Ahed cKO mice became anemic after E14.5, characterized by fetal liver atrophy, and died before birth. We analyzed the E14.5 liver of Ahed cKO fetuses to determine the significance of Ahed in fetal hematopoiesis. Flow cytometric analysis revealed that erythroid cells was significantly decreased in Ahed cKO livers. In contrast, a substantial number of lineage-Sca-1+c-KitHi (LSK) cells remained, whose phenotype corresponded to hematopoietic stem/progenitor cells (HSPCs). Transplantation experiments revealed that Ahed-deficient LSK cells could not reconstitute hematopoiesis in vivo. Collectively, these data determined that Ahed is indispensable for functional HSPCs. Based on its nuclear localization, we reasoned that the Ahed protein is functionally involved in intranuclear events. High-throughput RNA sequencing analyses of Ahed-cKO cells revealed that the protein governs various critical pathways by regulating alternative splicing of immature RNAs. Indeed, we confirmed that Ahed deficiency induces abnormal transcripts of critical genes for hematopoiesis. Importantly, published databases of cancer-related mutations present blood cancer patients with somatic mutations of the Ahed gene. Thus, Ahed is a newly discovered regulator essential for the integrity of hematopoiesis, whose mutations are likely to underlie hematopoietic malignancies. Summary/Conclusion: We have successfully identified a previously uncharacterised gene, Ahed, whose perturbation disrupts the development of hematopoiesis both in vitro and in vivo. The results shed light on the feasibility of in vitro differentiation screening assays using a mutant ESC bank. Since mutations of Ahed human orthologues were repeatedly found in cancer patients, further analyses of the molecular mechanism underlying their function might pave the way for novel cancer therapies. Keywords: Genetic, Alternative splicing, Hematopoietic stem and progenitor cells, Hematopoiesis